title: Enzymatic production of Cilastatin intermediate via highly enantioselective hydrolysis of methyl (±)-2,2-dimethylcyclopropane carboxylate using newly isolated Rhodococcus sp. ECU1013.
abstract: (S)-(+)-2,2-Dimethylcyclopropane carboxylic acid [(S)-(+)-DMCPA] is a key chiral intermediate for production of Cilastatin, an excellent renal dehydropeptidase-I inhibitor. In this study, a new method for preparation of (S)-(+)-DMCPA with microbial esterases was investigated. A microbial screening program obtained six esterase-producing isolates that could display relatively high activities and enantioselectivities using racemic ethyl 2,2-dimethylcyclopropane carboxylate (DMCPE) as screening substrate, aiming at forming optically pure (S)-(+)-DMCPA. Further selection was carried out with substrates having different alcohol moieties, including methyl, ethyl, and 2-chloroethyl esters. Finally, one of these strains, numbered ECU1013, with high enantioselectivity toward the hydrolytic resolution of methyl 2,2-dimethylcyclopropane carboxylate (DMCPM), afforded the (S)-product in 92 % ee, and was later identified as Rhodococcus sp. According to our research, there were several active esterases to DMCPM in cells of Rhodococcus sp. ECU1013; however, (S)-preferential esterase was selectively enriched based on the time-dependent profile of esterases biosynthesis, thereby the enantiomeric excess of biotransformation product (ee p) was constantly increased, finally maintained at 95 % (S). To improve the yield, various organic solvents were employed for better dispersion of the hydrophobic substrate. As a result, (±)-DMCPM of up to 400 mM in the organic phase of isooctane was enantioselectively hydrolyzed into (S)-(+)-DMCPA, with an isolation yield of 38 % and a further increase of ee p to 99 %.
authors.affiliation: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China. chaohongliu@126.com
authors.forename: Chao-Hong
authors.initials: CH
authors.lastname: Liu
authors.forename: Jiang
authors.initials: J
authors.lastname: Pan
authors.forename: Qin
authors.initials: Q
authors.lastname: Ye
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015377:Cilastatin / Q000378:metabolism*; D003521:Cyclopropanes / Q000737:chemistry / Q000378:metabolism*; D004950:Esterases / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010802:Phylogeny; D012240:Rhodococcus / Q000145:classification / Q000201:enzymology / Q000302:isolation & purification / Q000378:metabolism*; D012988:Soil Microbiology; D013237:Stereoisomerism
chemical_list: D001426:Bacterial Proteins; D003521:Cyclopropanes; D015377:Cilastatin; C417982:1,2-dimethylcyclopropane; D004950:Esterases